HIV-1 associated nephropathy (HIVAN) is a disease almost entirely limited to individuals of African descent. The pathogenesis of HIVAN is not fully understood, however dysfunction and injury to podocytes that form the glomerular blood filtration barrier are essential to the development of this disease. The mechanism of HIV-1- mediated podocyte injury is unclear mostly due to the lack of expression of classical HIV-1 entry receptors on these cells. However, findings that HIV-1 Nef recapitulates a HIVAN phenotype in experimental models, suggest that podocytes can be injured by Nef that is released by other productively infected cells. Further, striking racial disparities in the frequency of focal segmentl glomerulosclerosis (FSGS) and HIVAN have led to the discovery of genetic variants of the APOL1 gene that predispose African Americans homozygous for these risk alleles to the development of these nephropathies. However, the cellular function of the wild type (WT) APOL1 protein and the contribution of APOL1 risk mutants to podocyte damage in the face of HIV-1 infection remain unknown. This application looks at the APOL1 path that could be therapeutically exploited to protect against Nef-mediated podocyte injury in HIVAN. We show that APOL1 in podocytes strongly colocalizes with lysosomes and autophagosomes, suggesting that APOL1 may play a role in autophagy. We also demonstrate that APOL1 WT stimulates autophagy in differentiated podocytes. Since inheritance of the APOL1 gene risk variants is fully recessive, analysis of the effect of the APOL1 on autophagy will require a podocyte model homozygous for the APOL1 risk alleles. We have developed a method for isolation and culture of human podocytes from urine that will be used to explore the role of APOL1 in podocytes. In this application, we propose that wild type APOL1 is protective against HIVAN development by activating autophagic flux and counteracting the inhibitory effect of Nef on autophagy. In contrast, APOL1 risk variants lacking these functions permit the deleterious effects of Nef. We propose: 1) To dissect the mechanism(s) by which APOL1 and a high risk double mutant (APOL1 DM) contribute to autophagy initiation by comparing the interactions between these two molecules and the autophagy machinery, using immunoprecipitation (IP), western blotting and confocal microscopy. 2) To investigate the mechanism(s) by which APOL1 counteracts the suppression of autophagosome maturation by HIV-1 Nef, we will test whether APOL1 WT competes with Nef and thus stimulates autophagosome maturation. Binding partners of Nef and APOL1 in Beclin1/UVRAG/C-Vps complexes will be identified. Autophagosome fusion with lysosomes will be analyzed using a novel tandem fluorescent mRFP-EGFP-LC3 construct. Renal pathogenic SF2 Nef and mutants will be used to discern mechanism(s) by which SF2 Nef inhibits autophagy and how APOL1 WT overcomes Nef's inhibition. We believe that results of our proposal will lead to a novel therapeutic strategy aimed at preventing podocyte injury in HIVAN by stimulation of autophagic flux and by targeting Nef in the presence of APOL1 risk alleles.